.2022 Oct;113(10):3362-3375.

doi: 10.1111/cas.15485. Epub 2022 Aug 21.

Brca1^L63X^/+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer

Yuzuki Nakamura^{1 2}, Jo Kubota^{1 2}, Yukiko Nishimura¹, Kento Nagata¹, Mayumi Nishimura¹, Kazuhiro Daino¹, Atsuko Ishikawa¹, Takehito Kaneko^{3 4}, Tomoji Mashimo^{4 5}, Toshiaki Kokubo⁶, Masaru Takabatake^{1 2}, Kazumasa Inoue², Masahiro Fukushi², Masami Arai⁷, Mitsue Saito⁸, Yoshiya Shimada^{1 2}, Shizuko Kakinuma^{1 2}, Tatsuhiko Imaoka^{1 2}

Affiliations

¹ Department of Radiation Effects Research, National Institute of Radiological Sciences, Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology, Chiba, Japan.
² Department of Radiological Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, Japan.
³ Division of Fundamental and Applied Sciences, Graduate School of Science and Engineering, Iwate University, Morioka, Japan.
⁴ Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
⁵ Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
⁶ Laboratory Animal and Genome Sciences Section, Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology, Chiba, Japan.
⁷ Department of Clinical Genetics, Graduate School of Medicine, Juntendo University, Tokyo, Japan.
⁸ Department of Breast Oncology, Graduate School of Medicine, Juntendo University, Tokyo, Japan.

PMID:35851737
PMCID: PMC9530872
DOI: 10.1111/cas.15485

Brca1^L63X^/+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer

Yuzuki Nakamura et al. Cancer Sci.2022 Oct.

.2022 Oct;113(10):3362-3375.

doi: 10.1111/cas.15485. Epub 2022 Aug 21.

Authors

Affiliations

¹ Department of Radiation Effects Research, National Institute of Radiological Sciences, Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology, Chiba, Japan.
² Department of Radiological Sciences, Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, Japan.
³ Division of Fundamental and Applied Sciences, Graduate School of Science and Engineering, Iwate University, Morioka, Japan.
⁴ Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
⁵ Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
⁶ Laboratory Animal and Genome Sciences Section, Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology, Chiba, Japan.
⁷ Department of Clinical Genetics, Graduate School of Medicine, Juntendo University, Tokyo, Japan.
⁸ Department of Breast Oncology, Graduate School of Medicine, Juntendo University, Tokyo, Japan.

PMID:35851737
PMCID: PMC9530872
DOI: 10.1111/cas.15485

Abstract

Women who are heterozygous for deleterious BRCA1 germline mutations harbor a high risk of hereditary breast cancer. Previous Brca1-heterozygous animal models do not recapitulate the breast cancer phenotype, and thus all currently used knockout models adopt conditional, mammary-specific homozygous Brca1 loss or addition of Trp53 deficiency. Herein, we report the creation and characterization of a novel Brca1 mutant rat model harboring the germline L63X mutation, which mimics a founder mutation in Japan, through CRISPR-Cas9-based genome editing. Homozygotes (Brca1^L63X/L63X ) were embryonic lethal, whereas heterozygotes (Brca1^L63X/+ ) showed apparently normal development. Without carcinogen exposure, heterozygotes developed mammary carcinoma at a comparable incidence rate with their wild-type (WT) littermates during their lifetime. Intraperitoneal injection of 1-methyl-1-nitrosourea (25 or 50 mg/kg) at 7 weeks of age induced mammary carcinogenesis at comparable levels among the heterozygotes and their littermates. After exposure to ionizing radiation (0.1-2 Gy) at 7 weeks of age, the heterozygotes, but not WT littermates, displayed dose-dependent mammary carcinogenesis with 0.8 Gy^-1 excess in hazard ratio during their middle age; the relative susceptibility of the heterozygotes was more prominent when rats were irradiated at 3 weeks of age. The heterozygotes had tumors with a lower estrogen receptor α immunopositivity and no evidence of somatic mutations of the WT allele. The Brca1^L63X/+ rats thus offer the first single-mutation, heterozygous model of BRCA1-associated breast cancer, especially with exposure to a DNA break-inducing carcinogen. This implies that such carcinogens are causative and a key to breast cancer prevention in individuals who carry high-risk BRCA1 mutations.

Keywords: animal model; breast cancer; genome editing; hereditary breast and ovarian cancer syndrome; radiation carcinogenesis.

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Figures

**FIGURE 1**
Generation and heredity of theBrca1^L63X allele. (A) Structure of ratBrca1 and oligonucleotides used. Top, exon–intron structure. Middle, a magnified view. Bottom, nucleotide sequences. Arrowheads, primers; G1 and G2, guide RNAs; P1 and P2, probes. Uppercase, deoxyribonucleotides; lowercase, ribonucleotides; bold and underlined, location of the c.188T>A (p.L63X) mutation. (B) Sequence of cloned PCR products from the founder ratBrca1 exon 4. Arrows, induced mutation. (C–E) Heredity of theBrca1^L63X allele. (C) Standard curve of the allele‐specific quantitative PCR. ΔC_t, difference in the threshold cycles for the two molecular species. (D) Content of theBrca1^L63X allele in the genomic DNA of offspring (n = 40) ofBrca1^L63X/+ heterozygotes. Mean and SD (duplicate measurements). (E) Contingency table of observed and expected numbers of offspring with each genotype. (F) Representative photographs of uteri of pregnant dams at designated days postcoitum (dpc). Arrowheads, maldeveloping conceptuses. (G) Weight of individual conceptuses

**FIGURE 2**
Normal development ofBrca1^L63X/+ andBrca1^+/+ rats. (A) Body and organ weights ofBrca1^L63X/+ rats (L63X/+, blue) and WT littermates (+/+, orange). Data are shown as mean ± SD (n = 4–6). No significant difference suggested between genotypes or interaction between genotypes and age, whereas age‐related differences were significant for all measures (p < 0.001), by two‐way ANOVA. (B) Mammary gland development. Whole mounts of inguinal mammary glands are shown. I, day of postlactational involution; L, day of lactation; P, day of pregnancy; W, weeks of age. For pregnancy and lactation, rats were mated at 11 or 12 weeks of age. Scale bar in the bottom right panel applies to all

**FIGURE 3**
Spontaneous and 1‐methyl‐1‐nitrosourea (MNU)‐induced mammary carcinogenesis inBrca1^L63X/+ andBrca1^+/+ rats. (A) Kaplan–Meier plots depicting the incidence of palpable mammary carcinoma in the untreated groups. (B) Kaplan–Meier plots depicting the incidence of palpable mammary carcinoma in MNU‐treatedBrca1^L63X/+ andBrca1^+/+ rats.p values, log–rank test. HR, hazard ratio (95% confidence interval)

**FIGURE 4**
Radiation‐induced mammary carcinogenesis inBrca1^L63X/+ andBrca1^+/+ rats. (A,B) Kaplan–Meier plots depicting the incidence of palpable mammary carcinoma in (A)*Brca1*^L63X/+ and (B)*Brca1*^+/+ rats irradiated with 0.1–2 Gy at 7 weeks of age. Data for untreated groups (REF) are reproduced from Figure 3A. **p < 0.01 (compared with untreated, log–rank test). NS, not significant. (C–F) Cox regression–estimated hazard ratios (HR; dots) with nonirradiatedBrca1^+/+ rats as the reference, their 95% confidence interval (CI; error bars), and the fitted linear dose–response models (lines) during designated time periods. Numbers are linear coefficients with (95% CIs); number in parentheses in (F) indicates the upper limit of the 95% CI; *p < 0.05 (Cox regression). (G) Kaplan–Meier plot depicting the incidence of palpable mammary carcinoma in rats irradiated with 2 Gy at 3 weeks of age

**FIGURE 5**
Estrogen receptor α (ERα)‐positive rate of mammary carcinomas inBrca1^L63X/+ andBrca1^+/+ rats. (A, B) H&E stained tissue sections. (C, D) Immunostained sections for ERα. Brown staining indicates immunoreactivity. Carcinomas that developed in untreatedBrca1^L63X/+ (A,C) andBrca1^+/+ (B,D) rats. Scale bar in (A) applies to (A–D). (E,F) ERα‐positive rate of individual mammary carcinomas. Dots, individual data points; black horizontal line, mean; error bars, SD. *p < 0.05,t‐test

**FIGURE 6**
Analysis of somatic mutations of theBrca1 locus in the mammary carcinoma of theBrca1^L63X/+ rats. (A) Retention of heterozygosity as revealed by allele‐specific quantitative PCR. The occupancy of theBrca1^L63X allele was ~50% in normal kidney tissues and mammary carcinomas from rats that were unexposed (0 Gy) and irradiated (2 Gy) at 3 or 7 weeks of age. (B) Summarized results of the target sequencing of theBrca1 locus. MNU, 1‐methyl‐1‐nitrosourea;N, number of tumors analyzed. See Table S1 for detail

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References

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Brca1^L63X^/+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer

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Brca1^L63X^/+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer

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